We propose to identify and purify human hematopoietic stem cells with long-term engraftment capability. Initially, the major effort will be made to critically examine the dogma that the human hematopoietic stem cells are CD34+. This concept was seriously challenged by recent reports that murine hematopoietic stem cells with long-term engraftment capability are CD34- whereas the CD34+ cells provide short-term to mid- term engraftment in lethally irradiated mice. Using sheep/human xenograft model, we have obtained preliminary information that there are cells capable of long-term engraftment in the CD34-human marrow cell population. It is possible that human stem cells are primarily in the CD34- cell population. It is also possible that the human stem cells are in both CD34+ and CD34-cell populations. Alternatively, there may be a species difference in surface markers between human and murine hematopoiesis. Finally, this apparent controversy may be explainable on the basis of epitope differences of the anti-CD34 antibodies. We will clarify the status of CD34 expression by long-term and short- term engrafting cells and develop alternative methods for purification of human stem cells. We will also correlate the engraftment capabilities of the cell populations with the incidences of long-term culture-initiating cells (LTC-IC) and blast cell colony progenitors. Two types of xenograft assays will be used for quantitation of human engrafting cells-sheep/ human chimera and NOD/SCID mice. The sheep/human xenograft assay is particularly suited for the measurement of human long-term engrafting cells. NOD/SCID mouse model is more practical for limiting dilution studies. We already have exciting preliminary data regarding the use of myeloablated NOD/SCID newborn mice for quantitation of human engrafting cells.